A reliable method of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry for determining selenoamino acids in selenoproteins from Lactococcus lactis

Abstract

A reliable method for simultaneous determination of four organic selenium species by HPLC-ICP-MS was developed and implemented in determining organic selenoamino acids (Se-AAs) in selenoproteins from Lactococcus lactis (L. lactis) NZ9000. The method consisted of liberating Se-AAs from selenoproteins using ultrasound-assisted protease hydrolysis, and quantitatively detecting Se-AA speciations by HPLC-ICP-MS. After optimizations of proteolysis conditions, chromatographic conditions and determination conditions, the established method could efficiently separate the four Se-AAs, including SeCys, SeCys2, SeMeCys and SeMet within 10 min. It presented high sensitivity with the limits of detection and quantitation in the range of 0.197∼0.240 μg∙L^-1 and 0.788∼0.960 μg∙L^-1, respectively, good repeatability with a relative standard deviation (RSD) of less than 5%, and good recovery in the desired floating range of 90%∼105%, verifying the good accuracy. The method successfully detected four selenium species in the purified glutathione peroxidase (LlGPx) overexpressed in L. lactis NZ9000, SeCys (0.9716∼1.6784 μg∙g^-1), SeCys2 (1.0695∼1.2124 μg∙g^-1), SeMeCys (0.7288∼0.7984 μg∙g^-1) and SeMet (1.0058∼1.9571 μg∙g^-1), accounting for up to 80.14% of total selenium. There was no difference of order of magnitude in the four Se-AAs, indirectly indicating the random incorporation of selenium into selenoprotein LlGPx in L. lactis NZ9000. This work throws new light on the identification and biosynthesis of organic selenium species in selenoproteins and selenium-riched organisms like L. lactis.

Keywords: GPx; HPLC-ICP-MS; Selenium species; Selenoamino acids; Selenoprotein.